Sanger DNA sequencing

Through DNA sequencing we determine the exact order of the bases A, C, G and T in a DNA sample. Basically, the DNA is used as a template to generate a number of fragments that differ in length from each other with a single base. The generated fragments are sorted by their size. The bases at the end are identified, recreating the original DNA sequence.
The dideoxy (or the chain termination method) is the most used DNA sequencing method. It was perfected by Fred Sanger in late 70’s. The method works by using a modified base called the dideoxy base. Whenever a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, the dideoxy stops the replication reaction.

 

Basic principles

Four chemicals (represented by A, C, G, T Latin characters) are responsible of storing the information in the DNA molecule. The order of bases on a DNA strand is the DNA sequence.
Most DNA sequencing is carried out using the chain termination method. This involves the synthesis of new DNA strands on a single stranded template and the random incorporation of chain-terminating nucleotide analogues.
The chain termination method produces a set of DNA molecules differing by one nucleotide in length. The last base in each molecule can be identified by way of a unique label. Separation of these DNA molecules according to size places them in the correct order to read off the sequence.

 

How DNA sequencing works?

 

The DNA to be sequenced is provided in single-stranded form. This acts as a template upon which a new DNA strand is synthesised. The four nucleotides necessary for DNA synthesis, the enzyme DNA polymerase and a primer (a short sequence annealed to the template which initiates the new DNA strand) are constantly supplied during the DNA sequencing process. The nucleotides added to the growing DNA strand are complementary to those in the template strand.
Sequencing is achieved by including in each reaction a nucleotide analogue that cannot be extended and thus acts as a chain terminator. Four reactions are set up, each containing the same template and primer but a chain terminator specific for A, C, G or T. Because only a small amount of the chain terminator is included, incorporation into the new DNA strand is a random event. Each reaction therefore generates a collection of fragments, but every DNA strand will end at the same type of base (A, C, G or T).
The primers or nucleotides included in each of the four reactions contain different fluorescent labels allowing identification of the DNA strands terminating at each of the four bases. The reaction products are finally mixed and separated by gel electrophoresis, which separates DNA molecules according to size even if they differ in length by only a single nucleotide. As the DNA strands pass through a capillary tube, the fluorescent signal is detected and the base identified. The process can be automated by using DNA sequencing machines and last generation contig assembly software programs.

 

 

Applications

Lower-fidelity single-pass DNA sequencing is useful for the rapid accumulation of sequence data at the expense of some accuracy. Another application of DNA sequencing technology is resequencing the same DNA molecule over and over. This is necessary, for example, in the typing of single nucleotide polymorphisms.

Another obvious application of DNA sequencing technology is the accurate DNA sequencing of genes and genomes. If high accuracy is required each template is sequenced several times

 

Process automation

Using the Sanger technology, only about 1200-2000 bases can be sequenced in one run. This technique can generate sometimes DNA sequences whose ends are of questionable quality (the DNA sequence cannot be trusted). A human operator needs to manually inspect each sequence and cut the untrusted regions if necessary. Larger DNA molecules, including whole genomes, must be broken into smaller fragments before sequencing and then reassembled using dedicated software.
The process is called ‘sequence assembly’ or ‘contig assembly’ because the resulted DNA fragments are aligned together into a ‘contig’ that represents the original DNA sample. The process is very slow and involves hours, even days of repetitive work. We strongly recommend DNA Sequence Alignment because it is the only existing software that can automate the whole assembly process.

 

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Presenting DNA Sequence Alignment

the ultimate tool for DNA sequence assembly

DNA Sequence Alignment is an easy to use software package providing DNA and RNA sequence assembly functions (simple and batch), contig editing functions and chromatogram viewer/editor. We have created a fast assembly engine and a range of features that greatly speed up the process of sequence assembly and mutation analysis. It provides automated end trimming (clipping), so there is no need to trim manually the low quality bases from the ends of the trace files before contig assembly. This saves considerable time, especially when assembling many DNA sequences. DNA Sequence Alignment offers a smart navigation system that takes you to the location of each sequence ambiguity / mutation with only a single click. Our proprietary sequencing error correction algorithm provides further timesaving. The truly user-friendly interface makes DNA Sequence Alignment the best choice for DNA contig assembly.

This sequence aligner is ideal for assembly of all sequence types, including the SSU rRNA genes (16S and 18S rRNA), LSU rRNA genes (23S and 28S rRNA), protein genes, and/or genomic fragments. DNA Sequence Alignment is a valuable tool for studies of biodiversity, molecular ecology, sequencing of various functional and structural genes, or any application that is involved in the cloning and sequencing of DNA. It can read sequences in either TXT, FASTA, SEQ, ABI, or SCF file format. The DNA Sequence Alignment package is offered at a very affordable price ($299 for Student license/ $599 for Academic/ $699 for business), making it an ideal for universities, research institutions, or private companies performing DNA sequencing. Discounts apply if you purchase more than one license.

Starting with version 2.7, DNA Sequence Alignment can assemble contigs in batch mode. Now you can assemble all your sequences at once. Just define a pattern, press the start button and you are done. At the moment DNA Sequence Alignment is the only software on the market who offers batch contig assembly. Another new feature in version 2.7 is 'Assemble to reference'.